U.S. patent application number 13/578164 was filed with the patent office on 2012-12-06 for microscope and observation method.
This patent application is currently assigned to OSAKA UNIVERSITY. Invention is credited to Katsumasa Fujita, Shogo Kawano, Satoshi Kawata, Masahito Yamanaka.
Application Number | 20120307238 13/578164 |
Document ID | / |
Family ID | 44367556 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120307238 |
Kind Code |
A1 |
Fujita; Katsumasa ; et
al. |
December 6, 2012 |
MICROSCOPE AND OBSERVATION METHOD
Abstract
Provided is a microscope and an observation method which can
improve spatial resolution. A microscope according to an aspect of
the invention includes a laser light source (10), an objective lens
(16) that focuses light from the laser light source on a sample,
and a detector (22) that detects the laser light as signal light
from a sample (17) when the sample (17) is irradiated with the
laser light. The light is applied to the sample with an intensity
changed to obtain a nonlinear region where intensities of the light
and the signal light have a nonlinear relation due to occurrence of
saturation or nonlinear increase of the signal light when the light
has a maximum intensity, and the detector (22) detects the signal
light according to the intensity of the laser light to perform
observation based on a saturation component or a nonlinear increase
component of the signal light.
Inventors: |
Fujita; Katsumasa; (Osaka,
JP) ; Kawano; Shogo; (Osaka, JP) ; Yamanaka;
Masahito; (Osaka, JP) ; Kawata; Satoshi;
(Osaka, JP) |
Assignee: |
OSAKA UNIVERSITY
Osaka
JP
|
Family ID: |
44367556 |
Appl. No.: |
13/578164 |
Filed: |
February 8, 2011 |
PCT Filed: |
February 8, 2011 |
PCT NO: |
PCT/JP2011/000697 |
371 Date: |
August 9, 2012 |
Current U.S.
Class: |
356/301 ;
356/338; 356/425; 356/432; 356/445; 359/385 |
Current CPC
Class: |
G01N 2021/655 20130101;
G01N 2021/653 20130101; G02B 2207/114 20130101; G02B 21/002
20130101 |
Class at
Publication: |
356/301 ;
359/385; 356/338; 356/425; 356/432; 356/445 |
International
Class: |
G02B 21/14 20060101
G02B021/14; G01J 3/44 20060101 G01J003/44; G01N 21/59 20060101
G01N021/59; G01N 21/17 20060101 G01N021/17; G01N 21/47 20060101
G01N021/47; G01J 3/50 20060101 G01J003/50 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2010 |
JP |
2010-027838 |
Claims
1. A microscope comprising: at least one light source that emits
light; a lens that focuses light emitted from the light source and
irradiates a sample with the light; and at least one detector that
detects signal light generated by a multi-photon transition process
in the sample when the sample is irradiated with the light, wherein
the light is applied to the sample with an intensity changed to
obtain a nonlinear region in which an intensity of the light and an
intensity of the signal light have a nonlinear relation due to
occurrence of saturation or nonlinear increase generated by a
non-linear optical effect when the light has a maximum intensity,
the sample is irradiated with two light beams having different
wavelengths, the two light beams are modulated with different
modulation frequencies, and the detector detects the signal light
according to the intensity of the light, and the signal light is
demodulated with a frequency corresponding to one of a sum and a
difference between the modulation frequencies of the two light
beams to perform observation based on one of a saturation component
and a nonlinear increase component of the signal light.
2. A microscope comprising: at least one light source that emits
light; a lens that focuses light emitted from the light source and
irradiates a sample with the light; and at least one detector that
detects signal light generated by a multi-photon transition process
in the sample when the sample is irradiated with the light, wherein
the light is applied to the sample with an intensity changed to
obtain a nonlinear region in which an intensity of the light and an
intensity of the signal light have a nonlinear relation due to
occurrence of saturation or nonlinear increase generated by a
non-linear optical effect when the light has a maximum intensity,
the sample is irradiated with two light beams having different
wavelengths, the two light beams are modulated with the same
modulation frequencies and the same phase, the signal light from
the sample is generated by an m-photon reaction (m is a natural
number of 2 or more) of the two intensity-modulated light beams,
and the detector detects the signal light according to the
intensity of the light and extracts a (m+1) or higher-order
harmonic component for the modulation frequency to perform
observation based on one of a saturation component and a nonlinear
increase component of the signal light.
3. The microscope according to claim 1, wherein the detector
detects, as the signal light, scattered light generated by a
multi-photon transition, and generation of optical harmonics due to
other optical effects including the nonlinear optical effect causes
the signal light to be saturated.
4. The microscope according to claim 3, wherein the detector
detects at least one of hyper-Rayleigh scattering, Raman
scattering, stimulated Raman scattering, coherent anti-Stokes Raman
scattering, four wave mixing, stimulated emission, harmonics
generation, difference frequency generation, and sum frequency
generation, and signal light detected by the detector is separated
from the light by using a wavelength difference from the light
emitted from the light source.
5. A microscope comprising: at least one light source that emits
light; a lens that focuses light emitted from the light source and
irradiates a sample with the light; and at least one detector that
detects, as the signal light, at least one of reflection light,
transmitted light, and scattered light generated with the same
wavelength as that of the light from the light source upon
application of the light to the sample, wherein the light is
applied to the sample with an intensity changed to obtain a
nonlinear region in which an intensity of the light and an
intensity of the signal light have a nonlinear relation due to
occurrence of saturation generated by a non-linear optical effect
of the signal light when the light has a maximum intensity, and
generation of optical harmonics due to other optical effects
including a higher-order nonlinear optical effect causes the signal
light serving as at least one of the reflection light, transmitted
light, and scattered light to be saturated to perform observation
based on a saturation component of the signal light.
6. The microscope according to claim 5, further comprising a
modulator that performs intensity modulation such that the
intensity of the light changes according to time, wherein the light
is applied to the sample with an intensity where the signal light
has the nonlinear region at a peak time of the light, the modulator
performs intensity modulation and scans relative positions of the
light and the sample such that the relative positions are changed,
and the detector detects the signal light emitted from the sample,
and a harmonic component for a modulation frequency in the
modulator is extracted from the signal light detected by the
detector and is observed.
7. The microscope according to claim 6, wherein the signal light
emitted from the sample is generated by an n-photon reaction (n is
a natural number of 1 or more) of light, the intensity of the light
being modulated by the modulator, and a (n+1) or higher-order
harmonic component for the modulation frequency in the modulator is
extracted and observed.
8. The microscope according to claim 5, wherein intensity
modulation is performed such that the intensity of the light
changes according to time, the light source is a pulse light
source, and a repetition frequency of the pulse light source is
higher than the modulation frequency for the intensity
modulation.
9. The microscope according claim 5, wherein the intensity of the
light is changed such that the signal light is applied to the
sample with at least two intensities of a first intensity
corresponding to the nonlinear region and a second intensity
different from the first intensity, and one of a saturation
component and a nonlinear increase component of the signal light is
calculated based on the intensity of the signal light at the first
intensity and the intensity of the signal at the second
intensity.
10. The microscope according to claim 5, wherein the signal light
separated according to a wavelength difference is detected by a
plurality of the detectors.
11. An observation method that irradiates a sample with light and
observes the sample, the method comprising: focusing two light
beams having different wavelengths and irradiating the sample with
the light beams to generate signal light by a multi-photon
transition process in the sample; modulating intensities of the two
light beams with different modulation frequencies such that the
signal light from the sample has a nonlinear region in which an
intensity of the light and an intensity of the signal light have a
nonlinear relation due to occurrence of one of saturation and
nonlinear increase generated by a non-linear optical effect when
the light has a maximum intensity; detecting, by the detector, the
signal light according to the intensity of the light and
demodulating the signal light with a frequency corresponding to one
of a sum and a difference between the modulation frequencies of the
two light beams to perform observation based on one of a saturation
component and a nonlinear increase component of the signal
light.
12. An observation method that irradiates a sample with light and
observes the sample, the method comprising: focusing the light and
irradiating the sample with the light to generate, as the signal
light, at least one of reflection light, transmitted light, and
scattered light having the same wavelength as that of the light
from the light source; changing the intensity of the light such
that the signal light from the sample has a nonlinear region in
which an intensity of the light and an intensity of the signal
light have a nonlinear relation due to saturation of the signal
light generated by a non-linear optical effect when the light has a
maximum intensity; and generating optical harmonics due to other
optical effects including a higher-order nonlinear optical effect
to cause the signal light serving as at least one of the reflection
light, transmitted light, and scattered light to be saturated to
perform observation based on a saturation component of the signal
light.
13. The observation method according to claim 11, wherein the
sample is irradiated with the light in a state where a metallic
probe is placed near the sample with the light irradiation or in a
state where metal particles are added to the sample.
14. The observation method according to claim 13, wherein the
signal light is detected by scanning the metallic probe placed near
the sample with the light irradiation and irradiating the sample
with the light.
15. The microscope according to claim 2, wherein the detector
detects, as the signal light, scattered light generated by a
multi-photon transition, and generation of optical harmonics due to
other optical effects including the nonlinear optical effect causes
the signal light to be saturated.
16. The microscope according to claim 1, wherein intensity
modulation is performed such that the intensity of the light
changes according to time, the light source is a pulse light
source, and a repetition frequency of the pulse light source is
higher than the modulation frequency for the intensity
modulation.
17. The microscope according to claim 2, wherein intensity
modulation is performed such that the intensity of the light
changes according to time, the light source is a pulse light
source, and a repetition frequency of the pulse light source is
higher than the modulation frequency for the intensity
modulation.
18. The microscope according to claim 1, wherein the signal light
separated according to a wavelength difference is detected by a
plurality of the detectors.
19. The microscope according to claim 2, wherein the signal light
separated according to a wavelength difference is detected by a
plurality of the detectors.
20. The observation method according to claim 12, wherein the
sample is irradiated with the light in a state where a metallic
probe is placed near the sample with the light irradiation or in a
state where metal particles are added to the sample.
Description
TECHNICAL FIELD
[0001] The present invention relates to a microscope and an
observation method.
BACKGROUND ART
[0002] A spatial resolution of a general microscope is limited by
diffraction limit. Thus, existing microscopes have a problem that
once a light wavelength and a numeric aperture are determined, the
spatial resolution cannot be increased beyond a certain level.
[0003] The inventors of this application have proposed a
fluorescence microscope to improve the spatial resolution (Patent
Literature 1). In this fluorescence microscope, observation is
performed based on saturation components of fluorescence.
CITATION LIST
Patent Literature
[0004] [Patent Literature 1] International Patent Publication No.
WO 2006/061947
SUMMARY OF INVENTION
Technical Problem
[0005] However, the fluorescence microscope described above has a
problem that the spatial resolution for components other than
fluorescence cannot be improved.
[0006] The present invention has been accomplished in view of the
above-mentioned problem, and an object of the invention is to
provide a microscope and an observation method which can improve
spatial resolution.
Solution to Problem
[0007] A microscope according to a first aspect of the present
invention includes: at least one light source that emits light; a
lens that focuses light emitted from the light source and
irradiates a sample with the light; and at least one detector that
detects signal light generated by a multi-photon transition process
in the sample when the sample is irradiated with the light. The
light is applied to the sample with an intensity changed to obtain
a nonlinear region in which an intensity of the light and an
intensity of the signal light have a nonlinear relation due to
occurrence of saturation or nonlinear increase generated by a
non-linear optical effect when the light has a maximum intensity,
and the detector detects the signal light according to the
intensity of the light to perform observation based on one of a
saturation component and a nonlinear increase component of the
signal light. This enables observation based on one of saturation
and nonlinear increase of the signal light due to a nonlinear
optical loss.
[0008] According to a second aspect of the present invention, in
the microscope, the detector detects, as the signal light, at least
one of reflection light, transmitted light, and scattered light
having the same wavelength as that of the light from the light
source, and generation of optical harmonics due to other optical
effects including a higher-order nonlinear optical effect causes
the signal light to be saturated.
[0009] According to a third aspect of the present invention, in the
microscope, the detector detects, as the signal light, scattered
light generated by a multi-photon transition, and generation of
optical harmonics due to other optical effects including the
nonlinear optical effect causes the signal light to be
saturated.
[0010] According to a fourth aspect of the present invention, in
the microscope, the detector detects at least one of hyper-Rayleigh
scattering, Raman scattering, stimulated Raman scattering, coherent
anti-Stokes Raman scattering, four wave mixing, stimulated
emission, harmonics generation, difference frequency generation,
and sum frequency generation, and signal light detected by the
detector is separated from the light by using a wavelength
difference from the light emitted from the light source. This
enables observation with a high resolution using various types of
light.
[0011] According to a fifth aspect of the present invention, the
microscope further includes a modulator that performs intensity
modulation such that the intensity of the light changes according
to time. In the microscope, the light is applied to the sample with
an intensity where the signal light has the nonlinear region at a
peak time of the light; the modulator performs intensity modulation
and scans relative positions of the light and the sample such that
the relative positions are changed, and the detector detects the
signal light emitted from the sample; and a harmonic component for
a modulation frequency in the modulator is extracted from the
signal light detected by the detector and is observed. This enables
generation of one of saturation and nonlinear increase due to a
nonlinear optical loss with simplicity.
[0012] According to a sixth aspect of the present invention, in the
microscope, the signal light emitted from the sample is generated
by an n-photon reaction (n is a natural number of 1 or more) of
light, the intensity of the light being modulated by the modulator,
and a (n+1) or higher-order harmonic component for the modulation
frequency in the modulator is extracted and observed. This enables
generation of one of saturation and nonlinear increase without
increasing the intensity of the light incident on the sample in a
one-photon reaction and a multi-photon reaction.
[0013] According to a seventh aspect of the present invention, in
the microscope, the sample is irradiated with two light beams
having different wavelengths; the two light beams are
intensity-modulated with the same modulation frequency and the same
phase; the signal light emitted from the sample is generated by an
m-photon reaction (m is a natural number of 2 or more) of the two
light beams modulated by the modulator; and a (m+1) or higher-order
harmonic component for the modulation frequency is extracted and
observed. This enables generation of one of saturation and
nonlinear increase in a multi-photon reaction without increasing
the intensity of the light incident on the sample.
[0014] According to an eighth aspect of the present invention, in
the microscope, the sample is irradiated with two light beams
having different wavelengths; the two light beams are modulated
with different modulation frequencies; and the light beams are
demodulated with a frequency corresponding to one of a sum and a
difference between the modulation frequencies.
[0015] According to a ninth aspect of the present invention, in the
microscope, intensity modulation is performed such that the
intensity of the light changes according to time; the light source
is a pulse light source; and a repetition frequency of the pulse
light source is higher than the modulation frequency for the
intensity modulation.
[0016] According to a tenth aspect of the present invention, in the
microscope, the intensity of the light is changed such that the
signal light is applied to the sample with at least two intensities
of a first intensity corresponding to the nonlinear region and a
second intensity different from the first intensity; and one of a
saturation component and a nonlinear increase component of the
signal light is calculated based on the intensity of the signal
light at the first intensity and the intensity of the signal at the
second intensity. This enables observation based on one of a
saturation component and a nonlinear increase component with a
simple configuration.
[0017] According to an eleventh aspect of the present invention, in
the microscope, the signal light separated according to a
wavelength difference is detected by a plurality of the
detectors.
[0018] An observation method according to a twelfth aspect of the
present invention irradiates a sample with light and observes the
sample, the method including: focusing the light and irradiating
the sample with the light to generate signal light by a
multi-photon transition process in the sample; changing an
intensity of the light to obtain a nonlinear region in which an
intensity of the light and an intensity of the signal light have a
nonlinear relation due to occurrence of saturation or nonlinear
increase generated by a non-linear optical effect when the light
has a maximum intensity; detecting the signal light from the
sample; and performing observation based on one of a saturation
component and a nonlinear increase component of the signal light
detected. This enables observation based on one of saturation of
and nonlinear increase of the signal light due to a nonlinear
optical loss, thereby enabling observation with a high spatial
resolution without detecting harmonic generation.
[0019] According to a thirteenth aspect of the present invention,
in the observation method, the sample is irradiated with the light
in a state where a metallic probe is disposed near the sample or in
a state where metal particles are added to the sample. This enables
observation based on generation of one of saturation and nonlinear
increase due to a nonlinear optical loss and based on one of the
saturation and the nonlinear increase, with simplicity.
[0020] According to a fourteenth aspect of the present invention,
in the observation method, the signal light is detected by scanning
the metallic probe disposed near the sample and irradiating the
sample with the light. This enables observation based on generation
of one of saturation and nonlinear increase due to a nonlinear
optical loss and based on one of the saturation and the nonlinear
increase, with simplicity.
Advantageous Effects of Invention
[0021] According to the present invention, it is possible to
provide a microscope and an observation method which can improve a
spatial resolution.
BRIEF DESCRIPTION OF DRAWINGS
[0022] FIG. 1 is a diagram showing a configuration of a laser
microscope according to an embodiment of the present invention;
[0023] FIG. 2 is a graph schematically showing a spatial
distribution of scattered light and reflection light;
[0024] FIG. 3A is a graph schematically showing a change in
intensity of modulated laser light and scattered light generated by
irradiation of laser light;
[0025] FIG. 3B is a graph schematically showing a change in
intensity of modulated laser light and scattered light generated by
irradiation of laser light;
[0026] FIG. 4 is a graph schematically showing a relation between
the intensity of reflection light and the intensity of harmonics
with respect to laser light intensity;
[0027] FIG. 5 is a graph schematically showing a power spectrum of
reflection light intensity with respect to frequency;
[0028] FIG. 6A is a graph schematically showing a spatial
distribution of reflection light;
[0029] FIG. 6B is a graph schematically showing a spatial
distribution of first-order frequency components of reflection
light;
[0030] FIG. 6C is a graph schematically showing a signal obtained
by demodulating the intensity of reflection light by a second-order
harmonic frequency 2f.sub.m;
[0031] FIG. 7A is a graph schematically showing a power spectrum of
reflection light intensity at a spot center;
[0032] FIG. 7B is a graph schematically showing a power spectrum of
reflection light intensity at a spot end;
[0033] FIG. 7C is a graph schematically showing a power spectrum of
reflection light intensity between the center and an end;
[0034] FIG. 8 is a graph showing an optical transfer function
obtained when high-order modulation harmonic components are
detected;
[0035] FIG. 9A is a graph showing a relation between laser light
and scattered light;
[0036] FIG. 9B is a graph showing a relation between laser light
and second harmonics;
[0037] FIG. 9C is a graph showing a relation between incident light
and CARS light;
[0038] FIG. 9D is a graph showing a relation between incident light
and stimulated Raman scattered light;
[0039] FIG. 10 is a graph showing demodulated signals with respect
to incident light intensity;
[0040] FIG. 11 is a graph showing demodulated signals with respect
to incident light intensity;
[0041] FIG. 12 is a graph showing demodulated signals with respect
to incident light intensity;
[0042] FIG. 13 is a diagram showing point spread functions obtained
when a modulation range of incident light intensities is
changed;
[0043] FIG. 14 is a diagram showing a specific configuration
example of a laser microscope according to an embodiment of the
invention;
[0044] FIG. 15 is a graph showing nonlinear reflection
characteristics of a gold thin-film surface;
[0045] FIG. 16 is a graph showing nonlinear reflection
characteristics of an LBO crystal surface;
[0046] FIG. 17 is a graph showing nonlinear scattering
characteristics from gold fine particles; and
[0047] FIG. 18 is a graph showing saturation characteristics of
CARS light from L-Alanine.
DESCRIPTION OF EMBODIMENTS
[0048] Hereinafter, specific embodiments to which the present
invention is applied will be described in detail with reference to
the drawings. However, the present invention is not limited to the
embodiments described below. The following description and the
drawings are simplified as appropriate for clarity of
explanation.
First Embodiment
[0049] In this embodiment, a spatial resolution is improved by
utilizing saturation of reflection light, scattered light, or
transmitted light. That is, an observation of saturation components
of reflection light, transmitted light, or scattered light improves
a spatial resolution. A laser microscope according to a first
embodiment of the present invention is a confocal microscope, in
other words, a laser scanning type microscope. This laser
microscope is described with reference to FIG. 1. FIG. 1 is a
diagram schematically showing the configuration of the laser
microscope according to the present invention. Reference numeral 10
denotes a light source; 11, a modulator; 12, a beam splitter; 13, a
scanner; 14, a lens; 15, a lens; 16, an objective lens; 19, a
filter; 20, a lens; 21, a pinhole; 22, a detector; 23, a lock-in
amplifier; and 24, a processing apparatus. The laser microscope
shown in FIG. 1 detects reflection light (or scattered light).
[0050] The light source 10 is a laser light source that
continuously emits illumination light; for example, a
continuous-wave Ar ion laser or a semiconductor laser having a
wavelength in a visible range can be used. Here, the laser
wavelength is represented by .lamda.. Laser light serving as
illumination light is intensity-modulated with the modulator 11,
and takes a periodic function with a frequency f.sub.m. In this
example, intensity modulation is performed such that the intensity
of the laser light takes a cosine function. Assuming that f.sub.m
represents a frequency in intensity modulation;
.omega..sub.m(=2.pi.f.sub.m) represents an angular frequency;
[0051] and t represents time, the laser light intensity is
proportional to 1+cos(.omega..sub.mt). The intensity of the
intensity-modulated laser light takes a maximum value at
.omega..sub.mt=2n.pi. and a minimum value at
.omega..sub.mt=(2n+1).pi.(n is an arbitrary natural number). Note
that an initial phase is 0. In this example, light is modulated
with f.sub.m=100 kHz, for example. As the modulator 11, an
electro-optical modulator, an acousto-optical modulator, or the
like may be used.
[0052] The intensity-modulated laser light enters the beam splitter
12. The beam splitter 12 reflects a half of the incident light and
transmits the remaining half of the light. The laser light
transmitted through the beam splitter 12 enters the scanner 13. The
scanner 13 scans the laser light and changes the propagation
direction of the laser light, thereby changing the position of the
laser light on a sample 17. Note that the scanner 13 is used in the
above description, but a driving stage or the like may also be used
instead of the scanner 13. Instead of scanning with laser light,
the stage or the like on which the sample 17 is placed may be
driven. A combination thereof may also be used, as a matter of
course. For example, the scanner 13 may perform scanning in an
X-direction and the stage may perform scanning in a Y-direction.
That is, any configuration may be employed as long as scanning is
performed while changing a relative position between laser light
and the sample 17. Note that the scanning is not limited to
scanning only in two-dimensional directions. Scanning in three
dimensions may also be used.
[0053] The laser light scanned by the scanner 13 passes through the
lens 14 and the lens 15. The light refracted by the lenses 14 and
15 enters the objective lens 16. The objective lens 16 focuses the
laser light onto the sample 17 or into the sample 17. When the
laser light serving as illumination light enters the sample 17, the
illumination light is scattered or reflected. Accordingly, one or
both of scattered light and reflection light exit from the sample
17. Hereinafter, the light emitted from the sample 17 according to
laser light is referred to as "scattered light/reflection
light".
[0054] The scattered light/reflection light emitted from the sample
17 enters the objective lens 16. The scattered light/reflection
light is refracted by the objective lens 16 and enters the lens 15
and the lens 14. The scattered light/reflection light refracted by
the lenses 15 and 14 is descanned by the scanner 13. Then, the
scattered light/reflection light descanned by the scanner 13 is
reflected by the beam splitter 12 and enters the filter 19.
[0055] The filter 19 is an optical filter, for example, and splits
the light according to the wavelength. The filter 19 transmits the
wavelength of laser light and blocks optical harmonics. That is,
the filter 19 extracts only fundamental waves from the scattered
light/reflection light emitted from the sample 17. Accordingly, the
second or higher-order harmonics are blocked by the filter 19.
[0056] The scattered light/reflection light transmitted through the
filter 19 passes through the lens 20. The lens 20 enters the
pinhole 21. The pinhole 21 has a light transmission hole formed at
the center thereof to allow the scattered light/reflection light to
pass therethrough. That is, the light transmission hole of the
pinhole 21 is disposed on the optical axis. The objective lens 16,
the lens 15, the lens 14, and the lens 20 are arranged so that an
image formed on the sample 17 is focused on the pinhole 21. The
scattered light/reflection light having passed through the pinhole
21 enters the detector 22. The detector 22 measures the intensity
of the received scattered light/reflection light.
[0057] The scattered light/reflection light entering the detector
22 is based on the intensity-modulated laser light. The detector 22
is a sensor such as a photoelectron multiplier. This detector 22
outputs a detection signal to the lock-in amplifier 23 in
accordance with the intensity of the received light. The lock-in
amplifier 23 locks in a predetermined repetition frequency, and
lock-in detects a signal from the detector 22. Here, the lock-in
amplifier 23 receives a reference signal from the modulator 11, and
demodulates the signal with a frequency n times (n is an integer of
2 or more) higher than a modulation frequency f.sub.m of the
modulator 11. For example, assuming that the modulation frequency
f.sub.m is 100 kHz, the signal is demodulated with a frequency of
200 kHz, 300 kHz, . . . . As a result, high-order modulation
frequency components can be extracted and detected.
[0058] Further, the processing apparatus 24 controls the scanner
13, the modulator 11, and the lock-in amplifier 23 to perform
lock-in detection during the scanning operation of the sample 17.
In addition, the processing apparatus 24 forms an optical image
based on the signal output from the lock-in amplifier 23. In other
words, an optical image is formed based on the detected signal
during the scanning operation of the sample 17. The optical image
can be displayed on a screen or data on the optical image can be
stored by the processing apparatus 24 executing predetermined
operations. As a result, the optical image can be observed or
captured by scattered light or reflection light.
[0059] The laser microscope according to this embodiment configures
a confocal microscope. That is, the components are arranged such
that the laser light source 10 serving as a point light source is
in optically conjugate relationship with the sample 17, and the
sample 17 is in optically conjugate relationship with the pinhole
21. This enables detection of the scattered light/reflection light
through a confocal optical system. Hence, the spatial resolution
can be improved.
[0060] Next, the principle of high-resolution detection utilizing
saturation of light will be described with reference to FIGS. 2 to
4. In the following description, an example of detecting reflection
light will be described. FIG. 2 is a graph showing a spatial
distribution of intensities of laser light and reflection light. In
FIG. 2, the horizontal axis represents a position, and the vertical
axis represents light intensity. FIGS. 3A and 3B are graphs each
showing a change with time in intensity of intensity-modulated
laser light and reflection light. In FIGS. 3A and 3B, the
horizontal axis represents time, and the vertical axis represents
light intensity. Further, in FIG. 2 and FIGS. 3A and 3B, the solid
line represents laser light intensity, and the dashed line
represents reflection light intensity. For clarity of explanation,
in FIG. 2 and FIGS. 3A and 3B, the intensity of the reflection
light corresponds to the intensity of the laser light intensity
when the reflection light is not saturated. FIG. 4 is a graph
showing a relation between the laser light intensity and the
intensity of the light emitted from the sample.
[0061] In FIG. 4, the horizontal axis represents laser light
intensity, and the vertical axis represents signal light
intensity.
[0062] As shown in FIG. 2, the intensity of laser light reaches its
peak at the center of a spot (a position represented by x=a) and
decreases with distance from the spot center. In this embodiment,
the laser light intensity is modulated. Accordingly, the laser
light intensity changes with time at x=a as shown in FIG. 3A, and
the laser light intensity changes with time at x=b as shown in FIG.
3B. That is, since the intensity of the laser light is modulated,
the laser light changes in any position in accordance with a cosine
function. Here, x=a represents a peak position, and x=b represents
an off-peak position. Thus, the laser light intensity at x=a is
higher than the laser light intensity at x=b at any timing.
[0063] If the laser light intensity increases, the reflection light
is saturated. When the laser light intensity is extremely high, for
example, optical harmonics of the laser light are generated by a
nonlinear optical effect. That is, light having a wavelength that
is an integral multiple of a laser wavelength is generated. The
original laser light intensity is reduced by the amount of
generated optical harmonics, and a harmonic loss occurs. Note that
the term "harmonic loss" refers to a phenomenon in which the
intensity of signal light such as scattered light, transmitted
light, or reflection light from a sample is lost and saturated due
to a higher-order nonlinear optical effect or other optical
effects. Accordingly, this harmonic loss can be referred to as a
nonlinear optical loss (hereinafter referred to as a nonlinear
optical loss). That is, the intensity of the reflection light is
reduced by the amount of the generated nonlinear optical loss.
Hereinafter, the optical harmonics of laser light generated in the
sample are defined as laser harmonics. Assuming that .lamda.
represents a laser wavelength serving as a fundamental wave, the
wavelength of the laser harmonics is represented by n.lamda. (n is
a natural number of 2 or more). In the following description, the
optical harmonics of the reflection light may be defined as laser
harmonics. Further, in the following description, reflection light
having the same wavelength as that of laser light is
illustrated.
[0064] When the intensity of the laser light is low as shown in
FIG. 4, the laser light and the reflection light are proportional
to each other. However, as the intensity of the laser light
increases, the laser light and the reflection light are not
proportional to each other. Thus, even when the intensity of the
laser light is increased, the obtained reflection light is
saturated instead of increasing in proportion. That is, if there is
no saturation due to the nonlinear optical loss, the reflection
light intensity increases in proportion to the laser light
intensity as indicated by the dashed line of FIG. 4. In practice,
however, saturation occurs from a certain laser light intensity as
indicated by the solid line, and the reflection light intensity
hits a peak. Note herein that the phrase "saturation due to a
nonlinear optical loss" refers to a phenomenon in which the
relation between the laser light intensity and the reflection light
intensity deviates from the linear relation. That is, referring to
FIG. 4, at the reflection light intensity where the dashed line and
the solid line are spaced apart from each other, saturation of
reflection light components occurs. In other words, a saturation
region in which saturation of the reflection light occurs is a
nonlinear region in which the reflection light intensity has a
nonlinear relation with the laser light intensity. In the nonlinear
region, the reflection light having the same wavelength as the
laser wavelength is reduced by the amount of optical harmonics of
the reflection light.
[0065] Such saturation of the reflection light is more likely to
occur as the laser intensity increases. Further, as the laser
intensity increases, the saturation degree increases. Accordingly,
the laser light having the spatial distribution shown in FIG. 2 is
more likely to be saturated toward the peak position (x=a), and the
saturation degree of the reflection light is greatest at the peak
position. Further, the laser light that is intensity-modulated to
each cosine function as shown in FIGS. 3A and 3B is more likely to
be saturated during a time (.omega..sub.mt=2n.pi.) corresponding to
the vicinity of each peak, and the saturation degree of the
reflection light is greatest during the time corresponding to the
peak.
[0066] Assume herein that in the portion represented by x=b, the
reflection light is not saturated at any time and the laser light
having such an intensity that causes the reflection light to be
saturated in the vicinity of the peak position (x=a) is applied to
the sample 17. As a result, the reflection light having the
intensity distribution as indicated by the dashed line shown in
FIG. 2 is emitted. Specifically, referring to FIG. 2, in the
vicinity of the peak position (x=a), a difference in intensity
occurs between the laser light and the reflection light, and the
waveforms of the laser light and the reflection light substantially
match each other with distance from the peak position.
[0067] Further, the reflection light has an intensity as indicated
by the dashed line of FIG. 3A with respect to the
intensity-modulated laser light. That is, the intensity of the
laser light is high in the portion represented by x=a, so that the
reflection light is saturated. As shown in FIG. 3A, saturation of
the reflection light occurs at about the time
(.omega..sub.mt=2n.pi.) corresponding to each peak, but does not
occur at about the time (.omega..sub.mt=(2n+1).pi.) between peaks.
That is, in FIG. 3A, a difference in intensity between the laser
light and the reflection light occurs at about the time
corresponding to each peak, and the laser light and the reflection
light match each other after the time corresponding to each peak.
On the other hand, the intensity of the laser light is low in the
portion represented by x=b, so that the reflection light is not
saturated at any time. Accordingly, the laser light intensity and
the reflection light intensity are proportional to each other. FIG.
3B shows that these intensities match each other.
[0068] In the portion represented by x=b, the reflection light
intensity is proportional to 1+cos(.omega..sub.mt). On the other
hand, in the portion represented by x=a, the intensity of the
reflection light decreases at about the time corresponding to each
peak due to the saturation of the reflection light, so that the
reflection light intensity is not proportional to
1+cos(.omega..sub.mt). That is, in the portion represented by x=a,
second-order, third-order, . . . harmonic components (harmonics
with respect to the intensity modulation by the modulator 11)
appear in the reflection light intensity. The detection of these
harmonics enables extraction of information on only the spot center
of the laser light. That is, the harmonic components of the
intensity modulation include information on the spot center of the
laser light.
[0069] The intensity of the reflection light obtained when no
saturation occurs is proportional to the reflectance and the
incident light intensity. The intensity of the laser light is
expressed as B(1+cos (.omega..sub.mt)) and is proportional to
(1+cos(.omega..sub.mt)). Note that "B" represents an amplitude.
When saturation occurs, the reflection light intensity is
represented by a function of (1+cos(.omega..sub.mt)). Accordingly,
when the reflection light intensity is subjected to Taylor
expansion, the intensity can be expressed as power series of
(1+cos(.omega..sub.mt)). That is, the term of the n-th power of
(1+cos(.omega..sub.mt)) appears. Then, the term
cos(2.omega..sub.mt) appears in (1+cos(.omega..sub.mt)).sup.2 and
the term cos(3.omega..sub.mt) appears in (1+cos
(.omega..sub.mt)).sup.3. Thus, modulation harmonic components
appear. Note that the term "modulation harmonics" refers to
harmonics with respect to the intensity modulation of the laser
light intensity by the modulator 11, and are used to be
distinguished from optical harmonics of laser light, i.e., light
having a wavelength .lamda./n.
[0070] Here, when a spectrum for a modulation frequency is obtained
by performing Fourier transform on the reflection light intensity
indicated by the dashed line of FIG. 3A, an amplitude spectrum as
shown in FIG. 5 is obtained. FIG. 5 is a diagram schematically
showing the amplitude spectrum with respect to the modulation
frequency. Since the reflection light is represented by a periodic
function, the spectrum thereof is a line spectrum having a peak at
a predetermined frequency.
[0071] As described above, the modulation harmonic components
appear in the reflection light, so that peaks appear at positions
of f.sub.m, 2f.sub.m, 3f.sub.m, 4f.sub.m, . . . . Assuming herein
that the laser light has an amplitude B, the height of the peak at
a position nf.sub.m is proportional to B.sup.n. For example, the
peak at the position f.sub.m is proportional to B, and the peak at
the position 2f.sub.m is proportional to B.sup.2.
[0072] Thus, FIGS. 6A to 6C show a spatial distribution of the
reflection light having modulation harmonic components and a
spatial distribution of the modulation harmonic components. FIG. 6A
is a diagram showing a spatial distribution of signal light, which
is identical with the spatial distribution indicated by the dashed
line of FIG. 2. FIG. 6B shows first-order (.omega..sub.m)
components, and FIG. 6C shows second-order (2.omega..sub.m)
components (second-order modulation harmonic components). In FIGS.
6A to 6C, the horizontal axis represents a position and the
vertical axis represents light intensity. Note that the vertical
axes are illustrated on different scales in the drawings to clarify
the explanation.
[0073] The spatial distribution shown in FIG. 6B, which shows a
first-order component, is proportional to B. The spatial
distribution shown in FIG. 6C, which shows a second-order
component, is proportional to B.sup.2. Accordingly, as shown in
FIGS. 6B and 6C, the half-value width of the peak in the spatial
distribution of the second-order component is narrower than that of
the first-order component. Similarly, an n-order component is
proportional to B. Accordingly, higher-order components have a
narrower peak half-value width. In other words, the higher-order
components have a narrower peak width and a steeper peak.
Accordingly, if higher-order components are detected, each of
substantial reflection light spots can be expressed as a power of a
point spread function, which improves the spatial resolution. The
spatial resolution can be improved in proportion to the order of
the detected components. Note that FIG. 6A shows the sum of the
first-order component and all modulation harmonic components. The
spatial distributions shown in FIGS. 6B and 6C are based on the
saturation components of the reflection light. The spatial
distribution of the modulation harmonic components varies based on
the saturation components of the reflection light. That is, the
intensity of the modulation harmonic components varies depending on
the intensity of the saturation components of the reflection light.
Thus, the spatial resolution can be improved by observing the
sample based on the saturation components of the reflection
light.
[0074] As described above, the saturation degree varies depending
on the spatial position on the sample. That is, the saturation
degree at the spot center position (x=a) of the laser light is
large, and no saturation occurs at the spot end (x=b) of the laser
light. At the position between the spot end and the spot center,
the saturation degree is smaller than that at the spot center.
Accordingly, the amplitude spectrum for the modulation frequency
can be obtained by performing Fourier transform on the reflection
light intensity at each position as shown in FIGS. 7A to 7C.
[0075] FIG. 7A shows the amplitude spectrum at the spot center
(x=a). FIG. 7B shows the amplitude spectrum at the spot end (x=b).
FIG. 7C shows the amplitude spectrum at the position between the
spot center and the spot end. Thus, for example, the resolution can
be improved by performing observation according to the signal
components having the frequency 2f.sub.m. The observation may be
performed by focusing on the n-order component, as a matter of
course. The demodulation with a frequency n times higher than a
modulation frequency enables observation with a higher spatial
resolution.
[0076] FIG. 8 shows an example of an optical transfer function
obtained when higher-order modulation harmonic components are
detected. As shown in FIG. 8, the spatial resolution is increased
along with an increase in the order. Note that a combination with a
confocal detection method enables further improvement in the
spatial resolution. Thus, this embodiment can improve the spatial
resolution in all three dimensional directions. Note that a method
using a confocal detection method or two-photon response without
using the detection method of the present invention is
substantially the same as an optical transfer function of
2.omega..
[0077] For example, the detection of the second-order modulation
harmonic components enables detection with a spatial resolution
twice as high as that when the first-order frequency components are
detected. Thus, the detection of the second-order and third-order
modulation harmonic components can double or triple the spatial
resolution. This enables detection with a spatial resolution beyond
the diffraction limit and detection with a resolution higher than
that of the laser microscope of the related art. N-fold spatial
resolution can be attained by detecting the n-order component, as a
matter of course. Further, the reflection light having the laser
wavelength .lamda. is detected instead of the lost optical
harmonics. In other words, the detector 22 receives the reflection
light and detects the nonlinear optical loss. Thus, the detection
with a high S/N ratio can be attained.
[0078] Further, since the reflection light having the same
wavelength as that of the illumination light is detected, light
having the same wavelength as that of laser light may be detected
even in the case of observing the higher-order nonlinear effects.
That is, the detection of the fundamental wave enables observation
based on the nonlinear effects. Even when the wavelength of the
laser light is shortened, the detection can be carried out in a
typical optical system. For example, there is no need to propagate
light in an ultraviolet region through an optical system, thereby
enabling use of a lens, a mirror, and the like of general
specifications. In other words, since the nonlinear optical loss is
measured, a higher-order nonlinear response can be checked easily
without preparing an optical component or measurement instrument
for shorter wavelengths. Accordingly, a higher resolution can be
achieved while using an optical system for a visible range. The
separate detection of the nonlinear response enables
three-dimensional observation of the shape of the sample with a
spatial resolution beyond the diffraction limit.
[0079] As described above, the extraction and detection of n-order
(n is a natural number of 2 or more) components improves the
spatial resolution. Thus, the lock-in amplifier 23 performs lock-in
detection at a fixed predetermined frequency. Assume herein that
the detection frequency of the lock-in amplifier 23 is n times (n
is a natural number of 2 or more) higher than the modulation
frequency f.sub.m. Further, the lock-in amplifier 23 locks in a
phase at which the illumination light peaks to thereby perform
detection. This enables high-sensitivity detection as well as
detection of higher-order modulation harmonic components.
Accordingly, the spatial resolution can be further improved. Since
the detection frequency is an integral multiple of the modulation
frequency f.sub.m of laser light, electrically filtering the
frequency band enables extraction of the modulation harmonic
components with an extremely high sensitivity. Note that not only
the lock-in detection, but also the detection of modulation
harmonics using a high-pass filter can be attained.
[0080] Assume that the modulation frequency f.sub.m of the
modulator 11 is much higher than that for scanning of the sample
17. That is, the modulation frequency is increased such that a
plurality of peaks appears in a scanning time of an optical image
corresponding to one pixel. For example, when a scanning time
corresponding to one pixel of the optical image is 1 msec, a
modulation frequency is set to 100 kHz. In this case, 100 peaks of
the laser light appear in the scanning time of one pixel. The
modulation frequency is set higher than the scanning frequency of
the sample, thereby increasing the number of times of saturation
occurring in the scamming time of one pixel to perform detection
precisely.
[0081] According to the present invention, as the saturation degree
increases, the saturation can be easily detected and the resolution
can be improved. However, if the laser light intensity (density) is
increased to increase the laser light intensity, there is a
possibility that the sample is damaged. In this case, a pulse laser
light source is preferably used as the light source. In this case,
for example, a cosine function takes an envelope curve as indicated
by the dotted line of FIG. 3, and laser light has a pulse intensity
corresponding to the envelope curve. The use of the pulse laser
light source enables reduction in the total irradiation amount,
while achieving the light intensity (density) that reaches the
saturation. Hence, it is possible to prevent the sample from being
damaged. Assume herein that the repetition frequency of the pulse
laser light is set much higher than the modulation frequency. That
is, the frequencies are set such that a plurality of pulses appears
in one modulation period. For example, when the modulation
frequency is 100 kHz, a repetition frequency of 80 MHz can be used.
In this case, 800 pulses are included in one period. In this way,
precise detection is realized by setting the repetition frequency
of the pulse laser light higher than that of the modulation
frequency.
[0082] Note that any function other than the cosine function can be
used as the function for modulating the intensity of laser light,
as long as the function has a periodic function. Here, the laser
light intensity is set to an intensity at which saturation of
signal light occurs at the timing when the maximum laser light
intensity is obtained. That is, at the timing at which the maximum
laser light is obtained, the saturation of signal light occurs, and
the sample needs only to be irradiated with laser light in a
nonlinear range where the laser light and signal light have a
nonlinear relation.
[0083] The laser microscope according to this embodiment may also
have a configuration with no confocal optical system. That is, an
optical microscope other than a confocal microscope may also be
used by removing the pinhole 21. Since the intensity of laser light
from positions other than a focus position is low, the degree of
saturation of the signal light is small. That is, at positions
other than the focus position, the intensity of the laser light is
low and the signal light has the linear region which is not the
nonlinear region. Accordingly, the saturation components of the
signal light from positions other than the focus position are
reduced. This results in improvement of the resolution in a
Z-direction also in the configuration with no confocal optical
system. That is, the signal light has the linear region at a
position shifting from the focus position in the optical axis
direction.
[0084] Therefore, the saturation of the signal light does not
occur, which makes it possible to extract information only from the
focus position and its vicinity. This enables three-dimensional
observation with a simple configuration without using any confocal
optical system. Needless to say, the resolution can be further
improved by using a laser microscope including a confocal optical
system.
[0085] Though the reflection light is detected in the above
description, light transmitted through a sample may be detected. In
this case, the detector 22 is disposed on the opposite side of the
objective lens 16. Also in this case, laser harmonic components and
fundamental frequency components of the transmitted light are
separated using a wavelength difference. Furthermore, observation
based on scattered light (Rayleigh scattering, Mie scattering) may
also be carried out. Accordingly, the present invention can also be
achieved by optical microscopes having a configuration other than
the configuration shown in FIG. 1. That is, though the description
has been made using an epi-illumination type laser microscope in
FIG. 1, the present invention can also be applied to a
transmitted-light illumination type optical microscope. In the case
of measuring transmitted light or scattered light, the
above-described technique is used for the transmitted-light
illumination type optical microscope. The transmitted light and
scattered light may be detected simultaneously, as a matter of
course. Also in the case of performing observation based on
reflection light or scattered light, the above-described technique
is used for the epi-illumination type optical microscope.
Second Embodiment
[0086] In this embodiment, observation is performed based on
saturation components of signal light generated due to a nonlinear
optical effect. For example, scattered light or the like generated
in a sample due to the nonlinear optical effect is detected. In
this case, scattered light having a wavelength different from a
laser wavelength is detected. For example, various types of light
generated by hyper-Rayleigh scattering, harmonic generation, Raman
scattering, coherent anti-Stokes Raman scattering (CARS), four-wave
mixing, stimulated emission, difference frequency generation, sum
frequency generation, parametric fluorescence, or stimulated Raman
scattering (SRS), for example, may be detected. The scattered light
includes scattered light generated due to the nonlinear optical
effect. The generation of the nonlinear optical effect or other
optical effects causes a loss in light intensity, that is, a
harmonic loss. The term "harmonic loss" herein described refers to
a harmonic loss in signal light, which is a concept different from
a harmonic loss in laser light (fundamental wave). This harmonic
loss is a nonlinear optical loss (hereinafter referred to as
"nonlinear optical loss").
[0087] In the case of detecting scattered light or the like having
a wavelength different from that of incident laser light, the light
having the laser wavelength is prevented from entering the detector
22. In other words, the light having the laser wavelength is
separated from the laser harmonics by using a wavelength
difference. In this case, for example, an optical filter, such as a
band-pass filter, or a dichroic mirror is used. That is, in the
case of detecting scattered light (Raman scattering, hyper-Rayleigh
scattering, stimulated Raman scattering, coherent anti-Stokes Raman
scattering, harmonic generation, parametric fluorescence, four wave
mixing, stimulated emission, sum frequency generation, difference
frequency generation, etc.) having a wavelength different from the
laser wavelength, for example, the laser light (fundamental wave)
and the signal light are separated using a wavelength difference.
For example, an optical filter, such as a band-pass filter, or a
dichroic mirror is used. An optical element that blocks the laser
light is disposed between the detector and the sample. Note that in
the case of detecting scattered light, the light source 10 may be
disposed on the opposite side of the objective lens 16. That is,
the scattered light transmitted through the sample 17 may be
detected on the opposite side of the light source 10.
[0088] In this manner, the scattered light or the like from the
sample is detected by the detector 22 as signal light emitted from
the sample. Then, the observation may be performed using the
modulation harmonic components of the modulator 11. Also in this
embodiment, the modulation may be performed with second or
higher-order modulation frequency. Further, selection of a
frequency to be demodulated prevents the laser light intensity,
which causes saturation of the nonlinear optical loss, from
increasing. In this case, the frequency to be demodulated varies
depending on the type of light to be detected. For example, in the
case of the multi-photon reaction, the second-order component
(frequency 2f.sub.m) appears also in the linear region.
[0089] Accordingly, it is desirable to perform demodulation with
third or higher-order modulation harmonics. A preferable
demodulation frequency will be described below with reference to
FIGS. 9A to 9D.
(Regarding One-Photon Reaction)
[0090] First, FIG. 9A shows a relation between a laser light
intensity and a scatter light intensity in a one-photon reaction,
that is, a linear response, as illustrated in the first embodiment.
In FIG. 9A, the horizontal axis represents a laser light intensity
I.sub.ex and the vertical axis represents a scattered light
intensity Is.sub.cat. Note that the scattered light shown in FIG.
9A is scattered light having the same wavelength as the laser
wavelength. For example, Rayleigh scattered light or Mie scattered
light is output due to the one-photon reaction. That is, the
scattered light is obtained by a linear optical effect. When there
is no saturation, the laser light and scattered light linearly
change. Then, when the laser light intensity is increased,
saturation of the scattered light occurs. In this case, the
scattered light serves as a first-power response corresponding to
the first power of the laser light intensity to be modulated.
[0091] In the case of signal light generated by the linear optical
effect, the relation between the laser light and the signal light
is obtained as shown in FIG. 9A. That is, the scattered light,
reflection light, or transmitted light having the same wavelength
as the laser wavelength has a relation as shown in FIG. 9A. In this
case, as described above, the observation is performed using second
or higher-order modulation harmonics. For example, the detected
light detected by the detector 22 is demodulated with the frequency
2f.sub.m. In other words, the lock-in frequency of the lock-in
amplifier 23 is set to 2f.sub.m. Further, in this case, the signal
light having the same wavelength as the laser light wavelength is
detected and the light having a wavelength other than the laser
light wavelength is removed by the filter 19.
(Regarding Two-Photon Reaction)
[0092] On the other hand, FIG. 9B shows a relation between laser
light and signal light in a two-photon reaction which is a
nonlinear optical effect. FIG. 9B shows the case where the signal
light is generated by second harmonic generation (SHG). In FIG. 9B,
the horizontal axis represents the laser light intensity I.sub.ex
and the vertical axis represents a second harmonic intensity
I.sub.SHG. The second harmonic generation is a response due to a
two-photon reaction. That is, the second harmonic generation (SHG)
is a square response corresponding to the square of the laser light
intensity to be modulated. When there is no saturation, the square
of the intensity-modulated laser light (modulated light) and the
second harmonic (optical harmonics) linearly change. Then, when the
laser light intensity is increased, SHG saturation occurs. For
example, when the laser light intensity I.sub.ex is proportional to
(1+cos(.omega..sub.mt)) in the linear region in which the SHG
saturation does not occur, the SHG intensity I.sub.SHG is
proportional to (1+cos(.omega..sub.mt)).sup.2. When this expression
is developed, the following expression is obtained.
I.sub.SHG=1+2cos(.omega..sub.mt)+cos.sup.2(.omega..sub.mt) . . .
(1)
[0093] Note that in the above expression (1) and the following
expression (2), a proportional coefficient is set to "1" so as to
simplify the explanation. When cos.sup.2(.omega..sub.mt) is
transformed using cos(2.omega..sub.mt), the following expression
(2) is obtained.
I.sub.SHG=1+2cos(.omega..sub.mt)+(1+cos(2.omega..sub.mt))/2 . . .
(2)
[0094] Accordingly, in the case of the SHG, which is a square
response, non-saturation components (linear components) of the SHG
include the first-order component as well as the second-order
component. In the SHG, the first-order component (f.sub.m) and the
second-order modulation harmonic component (2f.sub.m) are present
for the modulation frequency (f.sub.m) of the laser light. That is,
the SHG intensity in the linear region also includes the
second-order modulation harmonic component.
[0095] In this case, the observation is performed using third or
higher-order components. For example, the detected light detected
by the detector 22 is demodulated with the frequency 3f.sub.m. In
other words, the lock-in frequency of the lock-in amplifier 23 is
set to 3f.sub.m. Further, in this case, signal light having a
wavelength twice as long as the wavelength of laser light is
detected, and the light having the same wavelength as that of the
laser light and the light of third or higher-order laser harmonics
are removed by the filter 19.
[0096] (Regarding Coherent Anti-Stokes Raman Scattering)
[0097] Next, an intensity I.sub.CARS of coherent anti-Stokes Raman
scattered light will be described with reference to FIG. 9C. In the
case of generating coherent anti-Stokes Raman scattered light, pump
light and Stokes light are applied. That is, two laser beams are
simultaneously applied to the same position of the sample by using
two laser light sources. The pump light has a wavelength different
from that of the Stokes light. Assume herein that Ip represents the
pump light intensity and Is represents the Stokes light intensity.
In this case, the CARS light intensity I.sub.CARS is proportional
to Ip.sup.2Is in the linear region in which the CARS light is not
saturated.
[0098] Accordingly, when the pump light intensity Ip is modulated
and the Stokes light intensity Is is not modulated, the Stokes
light intensity Is is constant. Thus, the square response is
obtained in which the CARS light intensity I.sub.CARS varies
depending on the square (Ip.sup.2) of the pump light to be
modulated. In this case, the observation is performed using third
or higher-order components, as in the case of second harmonics
(optical harmonics) described with reference to FIG. 9B. For
example, the detected light detected by the detector 22 is
demodulated with the frequency 3f.sub.m. In other words, the
lock-in frequency of the lock-in amplifier 23 is set to
3f.sub.m.
[0099] On the other hand, when the Stokes light intensity Is is
modulated without modulating the pump light intensity Ip, the pump
light intensity Ip is constant. Accordingly, the first-power
response is obtained in which the CARS light intensity I.sub.CARS
varies depending the first power (Is) of the intensity-modulated
Stokes light. Accordingly, the observation is performed using
second or higher-order modulation harmonics, as in the case of
scattered light described with reference to FIG. 9A. For example,
the detected light detected by the detector 22 is demodulated with
the frequency 2f.sub.m. In other words, the lock-in frequency of
the lock-in amplifier 23 is set to 2f.sub.m.
[0100] Needless to say, both the pump light intensity Ip and the
Stokes light intensity Is may be modulated. In this case, when the
pump light intensity Ip and the Stokes light intensity Is are
modulated with the same frequency f.sub.m, the observation is
performed using fourth or higher-order components (frequency
4f.sub.m). In the case of modulating both the pump light intensity
I and the Stokes light intensity Is with the same modulation
frequency, the intensities are preferably modulated at the same
phase. That is, it is preferable to match the timings at which two
light beams are applied to the sample with a maximum intensity.
[0101] Alternatively, the pump light intensity Ip and the Stokes
light intensity Is may be modulated with different frequencies.
When the intensities are modulated with different frequencies, the
intensities are demodulated with an integral multiple of a
frequency corresponding to the difference or sum between two
frequencies. For example, assuming that f.sub.m.sub.--.sub.p
represents the modulation frequency of the pump light and
f.sub.m.sub.--.sub.s represents the modulation frequency of the
Stokes light, the intensities are demodulated with a frequency n
(2f.sub.m.sub.--.sub.p-f.sub.m.sub.--.sub.s) corresponding to an
integral multiple of the difference or with a frequency n
(2f.sub.m.sub.--.sub.p+f.sub.m.sub.--.sub.s) corresponding to an
integral multiple of the sum.
(Regarding Stimulated Raman Scattering)
[0102] A stimulated Raman scattered light intensity I.sub.SRS will
be described with reference to FIG. 9D. In the case of generating
stimulated Raman scattered light, pump light and Stokes light are
applied. That is, two laser beams are simultaneously applied to the
same position of the sample by using two laser light sources. The
pump light has a wavelength different from that of the Stokes
light. Assume herein that Ip represents the pump light intensity
and Is represents the Stokes light intensity. In this case, the
stimulated Raman scattered light intensity I.sub.SRS is
proportional to IpIs in the linear region in which the signal light
is not saturated. Accordingly, in the case of modulating one of the
pump light intensity I and the Stokes light intensity Is, the
first-power response of the intensity-modulated laser light is
obtained. Accordingly, the observation is performed using second or
higher-order components, as in the case of scattered light
described with reference to FIG. 9A. Further, in the case of
modulating both the pump light intensity I and the Stokes light
intensity Is with the same modulation frequency, the square
response is obtained. Accordingly, the observation is performed
using third or higher-order modulation harmonics. In the case of
modulating both the pump light intensity I and the Stokes light
intensity Is with the same modulation frequency, the intensities
are preferably modulated at the same phase. That is, it is
preferable to match the timings at which two light beams are
applied to the sample with the maximum intensity.
[0103] Alternatively, the pump light intensity Ip and the Stokes
light intensity Is may be modulated with different modulation
frequencies. When the intensities are modulated with different
modulation frequencies, the intensities are demodulated with an
integral multiple of a frequency corresponding to the difference or
sum between two frequencies. For example, assuming that
f.sub.m.sub.--.sub.p represents the modulation frequency of the
pump light and f.sub.m.sub.--.sub.s represents the modulation
frequency of the Stokes light, the intensities are modulated with
the difference n (f.sub.m.sub.--.sub.p-f.sub.m.sub.--.sub.s) or the
sum n (f.sub.m.sub.--.sub.p+f.sub.m.sub.--.sub.s). Note that in the
case of modulating both the pump light intensity I and the Stokes
light intensity Is, the intensities are preferably modulated at the
same phase. Thus, in the case of making the two laser beams
incident, the intensities are demodulated with a frequency
corresponding to the sum or difference between an integral multiple
of a first modulation frequency and an integral multiple of a
second modulation frequency.
[0104] In this manner, the order of the demodulation frequency for
achieving the high-resolution observation is selected depending on
the order of the intensity-modulated laser light. In a nonlinear
optical reaction such as a multi-photon reaction, the
intensity-modulated laser light is desirably demodulated with an
order higher than the order at which the intensity-modulated laser
light contributes to the signal light intensity. For example,
assume that when the modulated laser light intensity is raised to
the n-th power, the linear region can be achieved. That is,
assuming that I.sub.m represents the modulated laser light
intensity and I represents the signal light intensity, the laser
light intensity is proportional to the n-power (I.sub.m.sup.n) of
I.sub.m in the linear region. In this case, the demodulation is
preferably performed with an order of (n+1) which is higher by one
order. Thus, the selection of the order for the demodulation
expands the observation technique according to the present
invention to the complicated multi-photon reaction, and prevents an
increase in the laser light intensity.
[0105] Specifically, when the intensity of only one laser beam is
modulated in a configuration for applying one laser beam or
applying two or more laser beams, the demodulation frequency is
selected depending on the order of the response for generating the
signal light (scattered light). That is, when the signal light from
the sample is generated by an n-photon reaction (n is a natural
number of 1 or more) of the intensity-modulated laser light, (n+1)
or higher-order modulation harmonic components for the modulation
frequency are extracted to thereby perform observation. For
example, in the case of the one-photon reaction, two or more
modulation harmonic components are preferably extracted, and in the
case of the two-photon reaction, three or more modulation harmonic
components are preferably extracted. This enables observation with
a high resolution.
[0106] Also in the fluorescence disclosed by Patent Literature 1,
the demodulation is performed with a third-order modulation
frequency in the case of detecting two-photon fluorescence, and the
demodulation is performed with a fourth-order modulation frequency
in the case of detecting three-photon fluorescence.
[0107] Also in the case of modulating the intensity of two or more
laser beams with the same modulation frequency in the configuration
for applying two or more laser beams, the demodulation frequency is
changed depending on the sum of the orders of responses for
generating the signal light (scattered light). That is, when the
signal light from the sample 17 is generated by an m-photon
reaction (m is a natural number of 2 or more) of two
intensity-modulated laser beams, (m+1) or higher-order modulation
harmonic components for the modulation frequency are extracted to
thereby perform observation. In other words, in the case of using
two laser beams, the demodulation is preferably performed with an
order greater than the number of photons (total number of photons)
of the two laser beams which contribute to the nonlinear optical
effect. This enables observation with a high resolution without
increasing the laser light intensity. For example, in the CARS
light, the two-photon reaction of pump light and the one-photon
reaction of Stokes light are obtained. In the case of detecting the
CARS light by modulating each of the pump light and the Stokes
light, a (2+1)=three-photon reaction is obtained. Accordingly, the
demodulation is preferably performed with a fourth or higher-order
modulation frequency. Note that in the case of modulating the
intensities of two laser beams and irradiating the sample with the
two laser beams, the intensity modulation is preferably performed
at the same phase.
[0108] Next, the saturation of the nonlinear optical loss will be
described. When the laser light intensity is set to an extremely
high level, saturation occurs also in the nonlinear optical loss
caused by the second harmonics (optical harmonics) shown in FIG. 4.
That is, the second harmonics (optical harmonics) themselves are
saturated. In this case, the nonlinear optical loss is generated
due to the third-order harmonics (optical harmonics) or high-order
optical harmonics. Accordingly, as the laser light intensity is set
to an extremely higher level, the second-order modulation harmonic
components decrease. Such a higher-order nonlinear optical loss
will be described with reference to FIG. 10.
[0109] FIG. 10 is a graph showing simulation results obtained by
calculating first-order to fourth-order components .omega..sub.m to
4.omega..sub.m in a three-photon reaction such as third harmonic
generation. In FIG. 10, the horizontal axis represents incident
light intensity and the vertical axis represents demodulated
signal, that is, signal light intensity for each modulation
harmonic component (including the first order). Saturation
components of the signal light appear in this demodulated signal.
In FIG. 10, the first-order to fourth-order components are
respectively represented by (Fundamental), (Second harmonic),
(Third harmonic), and (Forth harmonic).
[0110] In the case of the three-photon reaction, as described
above, a third-order component appears also in the linear region in
which the signal light is not saturated. Accordingly, in the linear
region, the second-order and third-order components increase at
substantially the same inclination according to the incident light
intensity. In the nonlinear region in which the saturation occurs,
the fourth-order component appears. A rise of the fourth-order
component is caused by the saturation of the second-order and
third-order components. The fourth-order component is also
saturated. In this case, a fifth-order or sixth-order component
appears. That is, the nonlinear optical loss of the fourth-order
component appears as the fifth-order or sixth-order component (not
shown in FIG. 10).
[0111] Furthermore, an inverse peak (for example, "A" point in FIG.
10) appears in the second-order component and the like in FIG. 10.
The term "inverse peak" refers to a point where the intensity
decreases steeply. In practice the inventors of this application
have found that the intensity rapidly decreases to the vicinity of
"0" at this inverse peak.
[0112] FIG. 11 shows demodulated signals obtained by changing the
scale. FIG. 11 shows the first-order to fourth-order components. As
shown in FIG. 11, the second-order to fourth-order modulation
components take negative values, and the phases are different for
each order.
[0113] Thus, the intensity of the modulation component greatly
varies depending on the intensity of the incident light (excitation
light). That is, even when the laser light intensity is increased,
the laser light intensity at which saturation of the modulation
harmonic component does not occur is present. Accordingly, a
saturation component of a certain order does not appear in the
laser light intensity corresponding to the inverse peak. Further,
at a certain laser light intensity, the phase of the modulation
harmonic component may be reversed and the modulation harmonic
component may take a negative value. Thus, when the laser light
intensity is set so as to include the intensity corresponding to
the inverse peak, the point spread function greatly varies.
[0114] Here, the inverse peak is set to be included in the range of
the laser light intensity to be modulated. In this case, the point
spread function greatly varies. Simulation results obtained by
calculating this point spread function will be described with
reference to FIGS. 12 and 13.
[0115] FIG. 12 is a graph showing demodulated signals with respect
to incident light intensity, and corresponds to FIG. 10. FIG. 12
shows the first-order to fourth-order components and the total
components. Note that certain five incident light values are
respectively represented by a, b, c, d, and e as indicated by
dashed lines and solid lines in the longitudinal direction of FIG.
12 to describe the range of the incident light intensity (laser
light intensity) to be modulated. As is seen from FIG. 12, the
incident light intensities increase in the order of d, e, c, b, and
a.
[0116] First, FIG. 13a shows the point spread function obtained
when the intensity modulation is performed such that the maximum
laser light intensity is set to "a". In this case, the modulation
range of the incident light intensity is "0" to "a". Note that FIG.
13a shows the point spread function of the first-order
component.
[0117] Next, a description is made of the case where the maximum
value of the laser light intensity is "b". In this case, the
modulation range of the incident light intensity is "0" to "b". The
point spread function of the third-order component is indicated by
"b" in FIG. 13. At the center position of the laser light spot, the
incident light intensity becomes the maximum "b". As shown in FIG.
12, the incident light intensity slightly smaller than the laser
light intensity "b" corresponds to the inverse peak of the
third-order component. Thus, at positions slightly apart from the
center position of the laser light spot, saturation of the
third-order component does not occur. In other words, at positions
slightly apart from the spot, the saturation of the third-order
component decreases. Accordingly, each point spread function in the
vicinity of the spot center takes a minimum value. The minimum
value shows a ring shape as shown in FIG. 13b.
[0118] Next, FIG. 13c shows the point spread function of the
third-order component obtained when the intensity modulation is
performed such that the incident light intensity has the maximum
value "c". Similarly, FIG. 13d shows the point spread function of
the third-order component obtained when the intensity is modulated
such that the incident light intensity has the maximum value "d".
As shown in FIGS. 13b to 13d, the third-order point spread function
varies depending on the modulation range. When the laser light
intensity is increased so as to obtain the modulation range
including the inverse peak, the point spread function of the
third-order component has a ring-shaped minimum value. The position
of the minimum value from the spot center varies depending on the
relation between the modulation range and the inverse peak. In this
manner, the point spread function varies depending on the
modulation range of the incident light intensity. In practice, an
image to be observed is represented by a combination of various
point spread functions. Accordingly, an image analysis is performed
from this point of view to thereby improve the resolution.
[0119] Next, FIG. 13e shows the point spread function of the
fourth-order component obtained when the intensity modulation is
performed such that the incident light intensity has the maximum
value "d". In this case, the minimum value is obtained at the spot
center, and the ring-shaped maximum value is present around the
spot center. Further, the ring-shaped minimum value is present
outside the ring-shaped maximum value. Accordingly, the point
spread function can be substantially reduced. This contributes to
an increase in the spatial resolution as compared to the
demodulation with the third-order modulation frequency.
[0120] Next, a specific configuration example of the
above-described laser microscope that detects saturation of signal
light will be described. FIG. 14 is diagram showing a configuration
for detecting transmitted light/scattered light transmitted through
a transparent sample 34 and reflection light/scattered light from
the sample 34. In FIG. 14, a pulse laser light source (omitted in
FIG. 14) having a repetition frequency of 80 MHz is used. In this
case, an ultra-short pulse laser light source having a wavelength
of 1200 nm is used as the light source. Needless to say, the laser
light source can be changed depending on the sample. The laser
light source is a mode-locked Ti:sapphire laser+OPO (light
parametric oscillator), and has a pulse width of, for example, 200
fsec. Further, nonlinear optical crystal such as BBO or LBO is used
as the sample 34. Note that in FIG. 14, the transmitted light is
detected by a non-confocal optical system. In the configuration
shown in FIG. 14, observation is performed using both the nonlinear
optical loss and the harmonic generation.
[0121] Then, a modulator 31 modulates the intensity of the laser
light. The modulator 31 is an AOM (acousto-optical modulator), for
example, and modulates the intensity with a single frequency (for
example, 2 MHz). The modulation frequency is set much lower than
the repetition frequency of the pulse of the laser light. The laser
light modulated by the modulator 31 enters a beam splitter 32. A
part of the light is transmitted through the beam splitter 32 and
the remaining light is reflected in the direction of a detector 39.
A signal output from the detector 39 is input to a lock-in
amplifier 40 as a reference signal. The laser light passing through
the beam splitter 32 is refracted by a lens 33 and enters the
sample 34.
[0122] The transmitted light/scattered light transmitted through
the sample 34 is refracted by an objective lens 35. The transmitted
light/scattered light enters a detector 41 through a dichroic
mirror 36. The detector 41 is a photodiode (PD), for example, and
receives light having the laser wavelength .lamda.. Further, the
dichroic mirror 36 reflects laser harmonics in the direction of a
mirror 37. Accordingly, the light having the wavelength .lamda./n
(n is a natural number of 2 or more) serving as laser harmonics is
reflected by the mirror 37 and enters a detector 42. The detector
42 is a photomultiplier (PMT) or a photodiode (PD), for
example.
[0123] The reflection light/scattered light reflected by the sample
34 enters the beam splitter 32 through the lens 33 serving as an
objective lens. Then, the reflection light/scattered light is
reflected in the direction of a line filter 43 by the beam splitter
32. The line filter 43 is a filter that allows the light having the
wavelength .lamda. to be transmitted, and blocks the light having
the wavelength .lamda./2 or smaller. This enables separation of the
signal light according to a wavelength difference.
[0124] The optical harmonics generated in the sample 34 are allowed
to propagate only in directions that satisfy phase matching
conditions.
[0125] Thus, even when optical harmonics are generated, signals may
be hardly detected depending on the angle between the crystal axis
of the sample 34 and the incident polarization direction (Moreaux,
JOSA B, 17 (2000) 1685). However, in the case of measuring a
harmonic loss, the propagation direction of the optical harmonics
has no effect on the measurement results. Since the polarization
direction of the incident light contributes only to the generation
efficiency of optical harmonics, an observation image that more
faithfully reflects the shape of the sample is obtained in the
microscope that measures the nonlinear optical loss. Thus, there is
a large difference in mechanism for forming an image contrast
between the case of detecting the nonlinear optical loss to create
an image and the case of detecting the optical harmonics generated
from a sample to create an image. Therefore, the detection of light
having the fundamental wave enables measurement of the nonlinear
optical loss.
[0126] A detector 38, the detector 39, the detector 41, and the
detector 41 output detection signals according to the intensity of
received light to the lock-in amplifier 40. These detection signals
are demodulated by the lock-in amplifier 40. Each of the detector
41 and the detector 38 measures a laser fundamental wave.
Specifically, the detector 41 detects scattered light/transmitted
light having the same wavelength as that of the laser light, and
the detector 38 detects scattered light/reflection light having the
same wavelength as that of the laser light. The detector 42
receives the transmitted light/scattered light through the dichroic
mirror 36 and the mirror 37, thereby detecting light having a
wavelength n times (n is an integer of 2 or more) higher than the
second harmonics, third harmonics, or the like of the laser light,
that is, the laser wavelength .lamda.. As a result, the generated
harmonics are detected. The detector 39 measures a change in
intensity of the laser light.
[0127] An upper limit of the order of each response to be detected
is determined by a signal-to-noise ratio in measuring the intensity
of the fundamental wave. In the measurement of the nonlinear
optical loss, an extremely large number of photons can be used as
signals because the laser light itself serves as signal light.
Accordingly, the measurement can be made using the whole dynamic
range of each detector, and the signal-to-noise ratio can be set to
an extremely high level. It is also effective to use lock-in
detection for the measurement of the nonlinear optical loss. A
typical photodiode has a detection dynamic range of about 100 dB,
which facilitates measurement of a higher-order nonlinear
response.
[0128] For example, when super-resolution observation of a sample
is performed using laser light having a wavelength of 400 nm, a
higher-order nonlinear response is generated only in a central
portion of a laser focal point. Therefore, a higher resolution can
be obtained by increasing the demodulation frequency. The sample 34
is not limited to the nonlinear optical crystal, but crystal,
semiconductor, and metal can also be used. Nanoparticles (having a
diameter of about tens to hundreds of nm) of each material are used
as the sample 34. The nonlinear optical loss generated on the
surface of the sample 34 is measured and displayed as an image.
Further, biomolecules (Campagnola, Nat. Biotechnol. 21 (2003) 1356,
Debarre, Nat. Method, 3 (2006) 47) that efficiently generates
optical harmonics, such as collagen, myosin, tubulin, and lipid,
can also be used. That is, material that efficiently generates
optical harmonics is preferably used as the sample 34.
[0129] In the case of applying laser light to metal, the reflection
light can be efficiently saturated by utilizing plasmon resonance
on the surface of the metal. That is, an electric field intensity
is enhanced due to plasmon resonance, so that a nonlinear optical
reaction is efficiently generated. In this case, metal particles or
a metallic probe may be disposed in the vicinity of the sample.
This allows the second harmonic generation, stimulated Raman
scattering, and coherent anti-Stokes Raman scattering to be
efficiently generated. In the case of detecting signal light while
scanning the metal particles or metallic probe, a near-field
optical microscope image can also be obtained with an improved
spatial resolution.
[0130] Furthermore, the plasmon itself is saturated. In this case,
the metal itself of the sample serves as a sample probe, like
fluorescent dye in fluorescence observation. Accordingly, a
harmonic loss occurs at a low laser intensity. This allows the
reflection light to be efficiently saturated. In the case of using
metal as a sample probe, metal particles are contained in the
sample to be observed.
[0131] Specifically, like in fluorescence labeling, metal particles
are added to the sample. Since metal is not discolored, unlike
fluorescence dye, the sample can be used for a long period of time
without causing the problem of lifetime, even when the laser light
intensity is increased. Incidence of a plasma resonance wavelength
having a metal microstructure allows the saturation to be generated
more efficiently.
EXAMPLE
[0132] FIGS. 14 to 17 show the actual measurement results. FIG. 14
is a graph showing nonlinear reflection characteristics of a metal
thin-film surface. In the measurement shown in FIG. 14, the laser
wavelength was set to 780 nm. FIG. 15 is a graph showing nonlinear
reflection characteristics of an LBO crystal surface. In the
measurement shown in FIG. 15, the laser wavelength was set to 780
nm. FIG. 16 is a graph showing nonlinear scattering characteristics
from gold fine particles.
[0133] In the measurement shown in FIG. 16, the laser wavelength
was set to 520 nm, and gold fine particles having a diameter of 50
nm were used. FIG. 17 is a graph showing saturation characteristics
of CARS light from L-Alanine. In the measurement shown in FIG. 17,
the wavelength of Stokes light was set to 820 nm and the wavelength
of pump light was set to 785 nm. Further, the Stokes light
intensity was set to 3100 kW/cm.sup.2 to be kept constant, and the
pump light was modulated.
[0134] As shown in FIGS. 14 to 17, in the region where the incident
light intensity was low, the incident light intensity and the
signal light intensity were proportional to each other. Meanwhile,
when the incident light intensity was increased, the signal light
intensity was saturated.
Other Embodiment
[0135] Though the laser light intensity is changed using a
modulator in the above description, the laser light intensity may
be changed by other methods. For example, as disclosed in Published
Japanese Translation of PCT International Publication for Patent
Application, No. 2006/061947, laser light is attenuated using a
filter such as an ND filter. The use of a filter allows the laser
light intensity to change stepwise. Further, when the laser light
intensity becomes maximum, saturation of the signal light is
generated. As a result, the same advantageous effects can be
obtained. For example, the intensity of the laser light is changed
such that signal light is applied to a sample with at least two
intensities, i.e., a first intensity at which the signal light has
the nonlinear region and a second intensity different from the
first intensity. Then, saturation components of the signal light
are calculated based on the intensity of the signal light at the
first intensity and the intensity of the signal light at the second
intensity.
[0136] Furthermore, various types of signal light may be detected
simultaneously by multiple channels. Specifically, multiple
detectors are provided and the detectors detect different types of
signal light. In this case, the signal light may be separated
according to a wavelength difference caused depending on the type
of the signal light.
[0137] Though the observation is performed based on the saturation
components of the signal light generated by the nonlinear optical
effect in the above description, the observation may also be
performed based on a nonlinear increase component of the signal
light generated by the nonlinear optical effect. For example, in
the case of using a saturable absorber as a sample or the like, a
nonlinear increase occurs in the signal light. That is, the laser
light and the signal light have no nonlinear relation, and the
signal light rapidly increases according to an increase in the
laser light intensity. In this case, the nonlinear increase
component of the signal light can be extracted by demodulation with
a higher-order modulation frequency. As a result, the same
advantageous effects can be obtained. Though the laser light source
that emits laser light is used in the above description, light
sources that emit light other than laser light can also be used.
Any type of light that is generated by a multi-photon transition
process may be used as the signal light.
[0138] This application is based upon and claims the benefit of
priority from Japanese patent application No. 2010-27838, filed on
Feb. 10, 2010, the disclosure of which is incorporated herein in
its entirety by reference.
INDUSTRIAL APPLICABILITY
[0139] The present invention is suitable for a microscope and an
observation method that detect and observe various types of light,
for example.
REFERENCE SIGNS LIST
[0140] 10 LIGHT SOURCE [0141] 11 MODULATOR [0142] 12 DICHROIC
MIRROR [0143] 13 SCANNER [0144] 14 LENS [0145] 15 LENS [0146] 16
OBJECTIVE LENS [0147] 17 SAMPLE [0148] 19 DICHROIC FILTER [0149] 20
LENS [0150] 21 PINHOLE [0151] 22 DETECTOR [0152] 23 LOCK-IN
AMPLIFIER [0153] 24 PROCESSING APPARATUS [0154] 31 MODULATOR [0155]
32 BEAM SPLITTER [0156] 33 LENS [0157] 34 SAMPLE [0158] 35
OBJECTIVE LENS [0159] 36 DICHROIC MIRROR [0160] 37 MIRROR [0161] 38
DETECTOR [0162] 39 DETECTOR [0163] 30 LOCK-IN AMPLIFIER [0164] 41
DETECTOR [0165] 42 DETECTOR
* * * * *